Use of phthalimide and/or sulphonamide derivatives in the treatment of diseases which require reducing the TNF-α levels and an exogenous source of nitric oxide, phthalimide derivatives, sulphonamide derivatives, and a method for obtaining a sulphonamide derivative

ABSTRACT

Preparation and use of phthalimide and/or sulphonamide derivatives with nitric oxide donor properties, having activities in increasing gamma-globin gene expression and anti-inflammatory and analgesic activities, effective in the treatment of hematologic diseases which require reducing the TNF-α levels and an exogenous source of nitric oxide, such as sickle-cell disease. The functionalized phthalimide derivatives are designed from the prototypes thalidomide and hydroxyurea.

This is a Divisional Application filed under 35 U.S.C. §120 as adivision of U.S. patent application Ser. No. 12/747,589, filed on Sep.7, 2010, which is a National Phase Application filed under 35 U.S.C.§371 as a national stage of PCT/BR2008/000386, filed on Dec. 12, 2008,an application claiming the benefit under 35 U.S.C. §119 of BrazilianApplication No. PI 0705396-7, filed on Dec. 12, 2007, the content ofeach of which is hereby incorporated by reference in their entirety.

The present invention describes the use of phthalimide derivatives withnitric oxide donor properties, which have important activities inincreasing the gamma-globin gene expression and anti-inflammatory andanalgesic activities, effective in the treatment of hematologic diseaseswhich require reduced TNF-α levels and an exogenous source of nitricoxide. More particularly, the present invention describes the use ofsuch phthalimide derivatives for the treatment of sickle-cell disease.

The Sequence Listing submitted in text format (.txt) on Jun. 14, 2012,named “30927UB_Sequence_Listing_(—)06122012.txt” (created on Jun. 12,2012, 3 KB), is incorporated herein by reference.

DESCRIPTION OF THE PRIOR ART

The sickle-cell disease is the most prevalent hematologic geneticdisease known, and is characterized by a point mutation in the β-globingene, more specifically a single nucleotide change (GTG into GAG) in thesixth codon of the β-globin gene, resulting in the substitution of aglutamic acid with valine on the surface of the β-globin chain variant(β^(S)-globin) (SAFO, M. K et al. J. Med. Chem. v. 47, pp. 4665-4676,2004).

The substitution of glutamate with a valine has major consequences onthe three-dimensional structure of hemoglobin. Glutamic acid isnegatively-charged and valine is a neutral amino acid, thereby allowingthe approximation of hemoglobin molecules, and, consequently, thepolymerization, when deoxygenized. In the deoxy conformation ofsickle-cell hemoglobin (Hb S), valine, which is present in the chain,carries out hydrophobic interactions with the pocket, comprised ofhydrophobic amino acids, from a neighboring Hb S molecule, which is notpossible in the oxygenated state of hemoglobin, since the hydrophobicpocket is inaccessible in this condition (ADACHI, K. et al. J. Biol.Chem. v. 263, n. 12, pp. 5607-5610, 1988).

These interactions lead to the polymerization of deoxy-Hb S at lowoxygen pressures, a typical situation of capillary beds inmetabolically-active tissues (AVILA, C. M. et al. Bioorg. Med. Chem. v.14, pp. 6874-6885, 2006).

The polymerization of Hb S is the central process of vaso-occlusion, acharacteristic of the sickle-cell disease (BUNN, H. F. N Engl J Med v.337, pp. 762-769, 1997; KAUL D. K. et al. Blood Rev. v. 10, pp. 29-44,1996; a) FERRONE, F. A. et al. J. Mol. Biol. v. 183, pp. 591-610, 1985.b) FERRONE, F. et al. J. Mol. Biol. v. 183, pp. 611-631, 1985; SAMUEL,R. E., et al. Blood. v. 82, pp. 3474-3481, 1993).

Due to the intracellular polymerization of hemoglobin, on account of theoxygenation-deoxygenation cycles, the cells containing Hb S take on asickle shape.

The sickling of red blood cells is associated with the reversiblechanges of the membrane. With repeated sickling/desickling cycles, theaberrations in the membrane function and structure become increasinglypronounced, culminating in the membrane being fixed in the sickled shape(LEE, G. R. et al. Vol. I Manole, 1998, pp. 1161-1163.)

The sickle red blood cells showed a normal adherence to the vascularendothelium, monocyte, and macrophages (DUITS, A. J. et al. Clin ImmunolImmunopathol v. 81, pp. 96-98, 1996; OKPALA, I. et al. J. Eur. J.Haematol. v. 69, pp. 135-144, 2002.)

This property of the sickle blood is given by the deformable sicklecells, but not by the irreversibly sickled cells, perhaps because therigid cells are not able to form multiple surface contacts with theendothelial cells. This fact denotes a strong positive correlation withthe frequency and severity of the pain crises. The turbulence areas inthe capillaries are the prevailing sites for adherence.

Vascular occlusion is the main event responsible for the clinicalpicture of sickle-cell disease, being the cause of pain crises and organfailure. Vaso-occlusive crises initiate at the venular microcirculation,as the sickle cells become trapped. The primary event that is criticalfor vaso-occlusion includes the adhesion of red blood cells(reticulocytes and deformed dense cells) to the venular endothelium.This adhesion leads to the formation of heterocellular aggregates (whiteblood cells and sickle cells), which also contribute to obstruction,resulting in local hypoxia, increase on formation of Hb S polymers, andpropagation of the occlusion of the neighboring vasculature. Neutrophiltransmigrations through endothelial gap junctions increase theinflammation in the microvasculature (OKPALA, I. et al. Eur. J.Haematol. v. 69, pp. 135-144, 2002.; OKPALA, I. Blood Rev. v. 18, pp.65-73, 2004).

Sickle red blood cell masses repeatedly clog the vessels of themicrocirculation, leading to painful vascular occlusion crises. 5% to10% among children or young adults with sickle-sell disease show, owingto the clogging of the microcirculation vessels, symptomatic pictures ofstroke, effusion or hemorrhage resulting from stenosis or aneurismaldilatation of important cerebral arteries.

It has been reported that the increase of fetal hemoglobin is beneficialto patients with sickle-cell disease, increasing its survival andreducing pain episodes (CHARACHE, S. et al. Blood. v. 79, pp. 2555-2565,1992).

Recently it has been reported that patients with sickle-cell diseaseshow a significant increase on the circulating levels of cytokines,including the tumor necrosis factor-alpha (MALAVÉ, I. et al. ActaHaematol. v. 90, pp. 172-176, 1993; FRANCIS, R. Jr., et al. J. Natl.Med. Assoc. v. 84: 611-615, 1992.; BUCHANAN et al. Hematology. pp.35-47, 2004), the increased expression of which is directly associatedwith different pathologies of inflammatory origin (MAKHATADZE, N. J.Hum. Immunol. v. 59, pp. 571-579, 1998).

TNF-α exerts pro-inflammatory effects, increasing the chemiotacticproperties, the adherence of neutrophils to the vascular endothelium,due to the increase of adhesion molecules, stimulating the production offree radicals and the synthesis of other inflammatory mediators, such asIL-1 and PGE2. TNF-α also induces changes on the coagulation andanticoagulation properties and increases the hepatic synthesis of someacute-phase reagents. Furthermore, it is an important mediator of septicsyndrome and endotoxic shock, being able to suppress the biosynthesis oflipoprotein lipases and lipogenic enzymes in adipose tissue, impairingthe storage of lipids on adipocytes.

The ability of TNF-α to change the anticoagulation properties of thevascular endothelium and to induce the pro-coagulation activity on thecellular surface of endothelium, stimulating the production of theplatelet-activating factor (PAF), and increasing the leukocyte adhesionto the vascular endothelium cells, results in a increase of theresistance to the blood flow, making circulation difficult and, thus,aggravating the microvascular stasis and the deoxygenation of Hb S.

Accordingly, an increase on the TNF-α blood levels in patients withsickle-cell disease may aggravate vaso-occlusive crises and also lead tothe occurrence of infectious and inflammatory episodes (MALAVÉ, I. etal. Acta Haematol. v. 90, pp. 172-176, 1993).

In fact, Malavé et al (MALAVÉ, I. et al. Acta Haematol. v. 90, pp.172-176, 1993), reported an interesting inverse correlation between thepercentage of fetal hemoglobin (Hb F) and the serum concentration ofTNF-α. These authors showed that patients having high plasma levels ofTNF-α exhibit a consequent reduction on Hb F levels. Taking into accountthat Hb F has a beneficial effect, improving the tissue oxygenation andreducing the polymerization of Hb S, such inverse correlation increasesthe risk of strokes concurrently with symptoms associated withsickle-cell disease. In addition, TNF-α has a major role on peripheralhyperalgesia, and its inhibition has been associated with the reductionof chronic and acute pain, which accounts for the analgesic effect ofthalidomide, the first anti-TNF-α drug introduced in therapeutics(RIBEIRO, R. A. et al. Eur. J. Pharmacol., v. 391, pp. 97-103, 2000).

For these reasons, the inhibition of TNF-α has been shown as animportant strategy for preventing vascular and inflammatorycomplications related with sickle-cell disease.

Various substances have been reported to have direct action on theinhibition of TNF-α. These substances include tumor necrosisfactor-alpha converting enzyme (TACE) inhibitors, neutralizingantibodies (infliximab), and drugs structurally related withthalidomide.

Many laboratories and research groups have been reported theanti-inflammatory and immunomodulatory properties of thalidomide,demonstrating its therapeutic potential against the treatment ofpathologies such as multiple myeloma, cachexia, tuberculosis, arthritis,among others (MIYACHI, H. et al. Bioorg. Med. Chem. v. 5, n. 11, pp.2095-2102, 1997.)

In this regard, the development of new thalidomide analogs, containingthe main pharmacophores for the inhibitory activity of TNF, and free oftoxicophoric moieties, responsible for the teratogenicity, constitutes aunique aim for the development of new therapeutic possibilities in thetreatment of pathologies associated with or aggravated by the increaseon the TNF plasma levels, as in the case of sickle-cell disease.

Nevertheless, there is no specific treatment for sickle-cell disease sofar. The treatment of this genetic disease is based on the use of drugswhich minimize or fight against the symptomatology of sickle-celldisease. Drugs which are useful to the symptomatic treatment availablein the market include desferrioxamine (Desferal®), anti-pneumococcalvaccines, prophylactic penicillin, folic acid (daily doses), andhydroxyurea (Hydrea®).

Hydroxyurea (HU) is a known inhibitor of the synthesis of ribonucleotidereductase, an enzyme responsible for converting ribonucleotides intodeoxyribonucleotides, interfering with DNA synthesis, and thus, limitsthe DNA synthesis (YARBRO, J. M. Semin. Oncol. v. 19, pp. 1-10, 1992;HANFT, V. N. et al. Blood. v. 95, n. 11, 3589-3593, 2000).

Although HU is the main drug available for the treatment of sickle-celldisease approved by the Food and Drug Administration (FDA) agency,various adverse effects are associated with its prolonged use, many ofwhich are due to its ability to interrupt the cell cycle in S and G1phases (BUCHANAN, G. R. et al. Hematology pp. 35-47, 2004; STUART, M. J.and NAGEL, R. L. Lancet. v. 364, pp. 1343-1360, 2004.), which actionscharacterize it as a cytotoxic and antineoplastic agent.

Recent studies have showed that therapy with hydroxyurea (HU) reducesdeaths associated with sickle-cell disease by 40%. The therapeuticbenefit of HU is based on the increase of the levels of fetal hemoglobin(Hb F), a genetically distinct hemoglobin that inhibits thepolymerization of deoxygenated sickle-cell hemoglobin (Hb S), preventingor hindering the occurrence of symptoms related with this pathology(STEINBERG, M. H. et al. JAMA, v. 289, pp. 1645-1651, 2003; CHARACHE, S.et al., Medicine v. 75, pp. 300-326, 1996).

Besides inhibiting the ribonucleotide reductase, HU also exerts itsaction mechanism as a nitric oxide (NO) donator drug, an importantmediator in maintaining the normal blood flow and pressure. It is knownthat HU reacts with oxy- and deoxyhemoglobin to form methemoglobin,which then reacts with another HU molecule in order to formiron-nitrosyl-hemoglobin (HbNO). The formation of HbNO involves a numberof reactions of the hydroxylamine moiety in order to form NO(COKIC, V.P. et al. Blood. v. 108, n. 1. pp. 184-191, 2006).

The benefit of nitric oxide (NO) in the treatment of sickle-cell diseaseis based on its ability to stimulate the production of fetal hemoglobin(Hb F) through the soluble guanylate cyclase (sGC) pathway. Theactivation of sGC increases the expression of γ-globin inerythroleukemic cells and primary human erythroblasts. The inhibition ofsGC prevents this increase, which suggests that the sGC pathwayregulates the expression of γ-globin, and consequently, the synthesis offetal hemoglobin (Hb F). Works have been demonstrating this hypothesis,showing that HU activates sGC and also induces the expression of mRNAγ-globin, increasing the levels of fetal hemoglobin (Hb F) in K562erythroleukemic cells and human progenitor cells (CONRAN, N. et al. Br.J. Haematol. v. 124, pp. 547-554. 2004).

These results suggest that the induction of Hb F mediated by NO inducesthe activation of sGC and support the therapeutic strategy based onnitric oxide for patients with sickle-cell disease.

Furthermore, NO has vasodilator effects, which aggregates beneficialeffects in physiopathology and in the treatment of sickle-cell disease.(KING, S. B. Free Rad. Biol. Med. v. 37, n 6, pp. 737-744, 2004).

The present invention relates to the novel use of some phthalimidederivatives and sulphonamide derivatives in the preparation ofalternative drugs for the treatment of diseases which involve the needof reducing the levels of the TNF-α factor and the need of an exogenoussource of nitric oxide. The invention described herein discloses asolution for the major limitations associated with the drug therapy ofdiseases which involve the need of reducing the levels of the TNF-αfactor and the need of an exogenous source of nitric oxide, providing analternative for the reduction of side and adverse effects ofcommonly-used compounds.

This invention also provides two new phthalimide derivatives which areused in the preparation of drugs for the treatment of said diseases, aswell as a new process for obtaining a specific sulphonamide derivativealso used in the preparation of drugs for the treatment of diseaseswhich involve the need of reducing the levels of the TNF-α factor andthe need of an exogenous source of nitric oxide. In a more particularaspect, the present invention overcomes the problems related with themajor limitations and complications associated with the drug therapyconventionally used for the treatment of sickle-cell disease, thusimproving the quality of life of the patient with sickle-cell disease.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides alternatives for the treatment ofdiseases in which there is an involvement of the increase of the TNF-αlevels and the need of an exogenous source of nitric oxide fortreatment.

In an aspect of the invention, the major limitations and complicationsassociated with the drug therapy usually employed in the treatment ofsickle-cell disease could be overcome or minimized with the use of thenitric oxide donor and TNF-α modulatory phthalimide and sulphonamidederivatives, thus improving the quality of life of the patient withsickle-cell disease.

The present invention refers to the use of a compound of general formula(I)

wherein W═H, halogen, NO₂, NH₂, OH, C₁-C₆ alcoxy, C₁-C₆ haloalcoxy,C₁-C₆ haloalkyl; R corresponds to C₁-C₇ alkyl, 2-phenyl, 3-phenyl,4-phenyl, 2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl, 3-ethylbenzyl,4-ethylbenzyl, benzyl, thiophene, furan, pyrrole, 2-pyridine,3-pyridine, 4-pyridine, pyrazine, pyrimidine, benzothiophene,benzofuran, indole, quinoline, isoquinoline, naphthalene,CH₂-2-thiophene, CH₂-3-thiophene, CH₂-2-furan, CH₂-3-furan,CH₃CH₂-2-thiophene, CH₃CH₂-3-thiophene, CH₃CH₂-2-furan, CH₃CH₂-3-furan;R′ corresponds to O—NO₂ ⁻ or SO₂NHOH or furoxan; or any pharmaceuticallyacceptable salt thereof, in the preparation of a drug for the treatmentof diseases which require reducing the levels of the TNF-α factor and anexogenous source of nitric oxide.

The invention also refers to the use of a compound of general formula(II)W—R₁—SO₂NHR₂  (II)wherein W═H, halogen, NO₂, NH₂, OH, C₁-C₆ alcoxy, C₁-C₆ haloalcoxy,C₁-C₆ haloalkyl, R₁ corresponds to 2-phenyl, 3-phenyl, 4-phenyl,2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl, 3-ethylbenzyl,4-ethylbenzyl, benzyl, thiophene, furan, pyrrole, 2-pyridine,3-pyridine, 4-pyridine, pyrazine, pyrimidine, benzothiophene,benzofuran, indole, quinoline, isoquinoline, naphthalene,CH₂-2-thiophene, CH₂-3-thiophene, CH₂-2-furan, CH₂-3-furan,CH₃CH₂-2-thiophene, CH₃CH₂-3-thiophene, CH₃CH₂-2-furan, CH₃CH₂-3-furan;R₂ corresponds to OH, H, C(═O)NHOH, C(═S)NHOH, C(═O)NOH(C₆H₅); or anypharmaceutically acceptable salt thereof, in the preparation of a drugfor the treatment of diseases which require reducing the levels of theTNF-α factor and an exogenous source of nitric oxide.

The invention still refers to a pharmaceutical composition for thetreatment of diseases which require reducing the levels of the TNF-αfactor and an exogenous source of nitric oxide comprising saidcomposition, said compound being selected among those resulting fromformulae I and/or II or combinations thereof in a pharmaceuticallyacceptable carrier.

The invention also refers to a method for obtaining the compound offormula IIA

comprising the following steps of:

a) mixing, in a suitable container, hydroxylamine hydrochloride, sodiumbicarbonate and water

b) adding ethanol to the mixture obtained in step a

c) adding 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzenesulphonylchloride to the mixture obtained in step b

The invention still refers to a compound of formula (IC):

and also to a compound of formula (IE)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Effect of derivatives (300 μmol/Kg), via i.p, in a mouse earedema assay induced by capsaicin. Values represent the mean and standarderror of the average of 5 animals. (*p<0.05 was considered significantat the 95% confidence level using Student's t test)

FIG. 2—Effect of derivatives (300 μMol/Kg), administered orally, in aperitonitis assay induced by 3% thioglycolate in mouse. Values representthe mean and standard error of the average of 4 animals. (*p<0.05 wasconsidered significant at the 95% confidence level using Student's ttest)

FIG. 3—Expression of gamma-globin mRNA in the presence of example 7 atdifferent concentrations, in the absence of hemin, at times of 24 h, 48h, 72 h and 96 h.

FIG. 4—Dose-response curve of compound IIA at concentrations of 5 μM, 30μM, 60 μM and 100 μM at times 24 h, 48 h, 72 h and 96 h in the absenceof hemin.

FIG. 5—Cell viability of the designed compounds.

FIG. 6—Dosing of nitric oxide by indirect pathway (nitrite)

DETAILED DESCRIPTION OF THE INVENTION

Currently, there is no specific treatment for hematologic diseases ofgenetic origin, but there are on the market drugs which are useful tothe symptomatic treatment, which improve the quality of life of patientsbearing these diseases.

The present invention has as its main novel characteristic the use offunctionalized phthalimide and/or sulphonamide derivatives in thepreparation of drugs for the treatment of diseases which require reducedlevels of the TNF-α factor and an exogenous source of nitric oxide. Theinvention also has as a novel characteristic the disclosure of newfunctionalized phthalimide derivatives designed from the prototypesthalidomide and hydroxyurea, and designed rationally through thestrategy of molecular hybridization for the treatment of said diseases.The invention also comprises, as another novel characteristic, a newmethod for obtaining a specific sulphonamide derivative which can beused in the preparation of a drug for the treatment of diseases whichrequire reducing the levels of the TNF-α factor and an exogenous sourceof nitric oxide.

The new derivative was obtained with good to excellent chemical yields,by employing a methodology characterized by having a few syntheticsteps, from commercially-available compounds, which qualifies thismethodology for industrial use.

The present invention refers to the use of a compound of general formula(I)

wherein W═H, halogen, NO₂, NH₂, OH, C₁-C₆ alcoxy, C₁-C₆ haloalcoxy,C₁-C₆ haloalkyl; R corresponds to C₁-C₇ alkyl, 2-phenyl, 3-phenyl,4-phenyl, 2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl, 3-ethylbenzyl,4-ethylbenzyl, benzyl, thiophene, furan, pyrrole, 2-pyridine,3-pyridine, 4-pyridine, pyrazine, pyrimidine, benzothiophene,benzofuran, indole, quinoline, isoquinoline, naphthalene,CH₂-2-thiophene, CH₂-3-thiophene, CH₂-2-furan, CH₂-3-furan,CH₃CH₂-2-thiophene, CH₃CH₂-3-thiophene, CH₃CH₂-2-furan, CH₃CH₂-3-furan;R″ corresponds to O—NO₂ ⁻ or SO₂NHOH or furoxan; or anypharmaceutically-acceptable salt thereof, in the preparation of a drugfor the treatment of diseases which require reducing the levels of theTNF-α factor and an exogenous source of nitric oxide. Preferably, thecompound of general formula I described is used in the preparation of adrug for the treatment of sickle-cell disease.

In a preferred embodiment of the invention, the compound designated by(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl nitrate is used in thepreparation of a drug for the treatment of diseases which requirereducing the levels of the TNF-α factor and an exogenous source ofnitric oxide. Said compound has the structural formula (IA):

and is preferably used in the preparation of a drug for the treatment ofsickle-cell disease.

In another preferred embodiment of the invention, the compounddesignated by 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl nitrate isused in the preparation of a drug for the treatment of diseases whichrequire reducing the levels of the TNF-α factor and an exogenous sourceof nitric oxide. Said compound has the structural formula shown asfollows (IB):

and is preferably used in the preparation of a drug for the treatment ofsickle-cell disease.

Another preferred embodiment of the invention uses the compounddesignated by 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitratein the preparation of a drug for the treatment of diseases which requirereducing the levels of the TNF-α factor and an exogenous source ofnitric oxide. Said compound has the structural formula (IC):

and is preferably used in the preparation of a drug for the treatment ofsickle-cell disease.

In still another preferred embodiment of the invention, the compounddesignated by 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitrateis used in the preparation of a drug for the treatment of diseases whichrequire reducing the levels of the TNF-α factor and an exogenous sourceof nitric oxide. Said compound has the structural formula (ID):

and is preferably used in the preparation of a drug for the treatment ofsickle-cell disease.

In another preferred embodiment of the invention, the compounddesignated by 2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethylnitrate is used in the preparation of a drug for the treatment ofdiseases which require reducing the levels of the TNF-α factor and anexogenous source of nitric oxide. Said compound has the structuralformula (IE):

and is preferably used in the preparation of a drug for the treatment ofsickle-cell disease.

The present invention also refers to the use of a compound of generalformula (II)W—R₁—SO₂NHR₂  (II)wherein W═H, halogen, NO₂, NH₂, OH, C₁-C₆ alcoxy, C₁-C₆ haloalcoxy,C₁-C₆ haloalkyl, R₁ corresponds to 2-phenyl, 3-phenyl, 4-phenyl,2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl, 3-ethylbenzyl,4-ethylbenzyl, benzyl, thiophene, furan, pyrrole, 2-pyridine,3-pyridine, 4-pyridine, pyrazine, pyrimidine, benzothiophene,benzofuran, indole, quinoline, isoquinoline, naphthalene,CH₂-2-thiophene, CH₂-3-thiophene, CH₂-2-furan, CH₂-3-furan,CH₃CH₂-2-thiophene, CH₃CH₂-3-thiophene, CH₃CH₂-2-furan, CH₃CH₂-3-furan;R₂ corresponds to OH, H, C(═O)NHOH, C(═S)NHOH, C(═O)NOH(C₆H₅); or anypharmaceutically-acceptable salt thereof, in the preparation of a drugfor the treatment of diseases which require reducing the levels of theTNF-α factor and an exogenous source of nitric oxide. Preferably, thecompound of general formula previously described is used in thepreparation of a drug for the treatment of sickle-cell disease.

In a preferred embodiment of the invention, the compound designated by4-amino-N-hydroxybenzenesulphonamide is used in the preparation of adrug for the treatment of diseases which require reducing the levels ofthe TNF-α factor and an exogenous source of nitric oxide. Said compoundhas the structural formula (IIA):

and is preferably used in the preparation of a drug for the treatment ofsickle-cell disease.

The subject invention still refers to pharmaceutical compositions forthe treatment of diseases which require reducing levels of the TNF-αfactor and an exogenous source of nitric oxide comprising saidcompositions, said compounds being selected among those resulting fromformulae I and/or II or combinations thereof. The compositions describedin the subject invention comprise compounds of general formula I and/orII or pharmaceutically acceptable salts thereof, in association with apharmaceutically acceptable excipient.

The pharmaceutical compositions of the present invention may beadministered in a variety of dosage forms, such as orally, in the formof tablets, capsules, sugar or tablets covered with a film, liquidsolutions or suspensions; rectally in the form of suppositories;parenterally, i.e., intramuscularly, or by infusion or intravenousand/or intrathecal and/or intraspinal injection.

The pharmaceutical compositions to which this invention relates areusually prepared according to conventional methods and administered in asuitable pharmaceutical form.

Solid oral pharmaceutical forms may contain, together with the activecompound, different diluents, such as lactose, dextrose, saccharose,cellulose, corn starch, potato starch, or other suitable diluents;lubricants, such as silica, talc, stearic acid, magnesium or calciumstearate, and/or polyethylene glycols or other pharmaceuticallyacceptable lubricants; binding agents such as starches, gum arabic,gelatin, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone,or other suitable binding agents; disaggregating agents, such as starch,alginic acid, alginates or starch or sodium glycolate, or other suitabledisaggregating agents; effervescent mixtures; dyes; sugary materials;wetting agents such as lectin, polysorbates, laurylsulphates; and,generally, non-toxic pharmacologically inactive substances used inpharmaceutical formulations. Preparations of said pharmaceuticalcompositions may be performed in a known way, such as by means ofmixture, granulation, pressing into tablets, sugar covering, filmcoating processes or other suitable processes.

Liquid dispersions for oral administration may include, for example,syrups, emulsions and suspensions. Syrups may contain, as a carrier, forexample, saccharose or saccharose with glycerine and/or mannitol and/orsorbitol or another pharmaceutically acceptable carrier. Suspensions andemulsions may contain, as a carrier, among others, a natural gum, agar,sodium alginate, pectin, methylcellulose, carboxymethylcellulose,polyvinyl alcohol or other suitable carriers.

Suspensions or solutions for intramuscular injection may contain,together with the active compound, a pharmaceutically acceptablecarrier, i.e., sterile water, olive oil, ethyl oleate, glycols, i.e.,polyethylene glycol, or other pharmaceutically acceptable carrier, and,if desired, a suitable amount of lidocaine hydrochloride. Solutions forintravenous injections or infusions may contain, as a carrier, forexample, sterile water, or preferably, they may be in the form ofsterile salt, aqueous or isotonic solutions, or may contain, as acarrier, propylene glycol or another pharmaceutically acceptablecarrier.

The suppositories may contain, together with the active compound, apharmaceutically acceptable carrier, such as cocoa butter, polyethyleneglycol, sorbitan polyoxyethylene, fatty acid ester surfactant, lecithin,or other pharmaceutically suitable carriers.

The present invention also refers to a novel method for obtaining thecompound designated by 4-amino-N-hydroxybenzenesulphonamide of formula(IIA)

through the following steps of:

-   -   a) mixing, in a suitable container, hydroxylamine hydrochloride,        sodium bicarbonate and water    -   b) adding ethanol to the mixture obtained in step a    -   c) adding        4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzenesulphonyl        chloride to the mixture obtained in step b

In a preferred embodiment of the invention, the synthesis of4-amino-N-hydroxybenzenesulphonamide is carried out by adding, into a 10mL round-bottom flask, 21.6 mg of hydroxylamine hydrochloride (0.31mmol), 26.1 mg of sodium bicarbonate (0.31 mmol), and 0.1 ml ofdistilled water. Upon ceasing the elimination of CO₂, 2 mL of ethanolwas added. Next, 100 mg of4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzenesulphonyl chloride(0.31 mmol) were added. The reaction was observed by thin layerchromatography (TLC) (eluent: 100% Dichloromethane) until the end ofreaction was indicated. After 45 minutes under reaction, the solvent isevaporated under reduced pressure, and the obtained product is washedwith hot dichloromethane (so as not to remove the4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzenesulphonyl chloridewhich did not react in order to give approximately 82 mg (80%) of4-amino-N-hydroxybenzenesulphonamide as a white powder, with a meltingrange higher than 275° C. (C₁₄H₈ClNO₄S; PM=318.306). The compoundobtained (4-amino-N-hydroxybenzenesulphonamide) through the describedmethod is used, as detailed previously, in the treatment of diseaseswhich require reducing the levels of the TNF-α factor and an exogenoussource of nitric oxide. Preferably, the obtained compound(4-amino-N-hydroxybenzenesulphonamide) is used in the preparation of adrug for the treatment of sickle-cell disease.

The present invention still refers to a new phthalimide derivativedesignated by 3-(1-3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitraterepresented by the formula shown as follows (IC)

and used, as described previously, in the preparation of a drug for thetreatment of diseases which require reducing the TNF-α levels and anexogenous source of nitric oxide.

The invention also refers to another phthalimide derivative designatedby 2-[4-(1-3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate ofgeneral formula shown as follows (IE)

and used, as described previously, in the preparation of a drug for thetreatment of diseases which require reducing the TNF-α levels and anexogenous source of nitric oxide.

The compounds described in the present invention were subjected to anumber of tests in order to ensure the intended activities in thetreatment of diseases which require reducing the TNF-α levels and anexogenous source of nitric oxide. Particularly, the compounds weresubjected to tests in order to ensure their activity as auxiliary agentsin the treatment of symptoms of sickle-cell disease. The tests carriedout and the results obtained are described in the following.

Mutagenic Activity Test

Firstly, the compounds were evaluated with the AMES test in order toidentify a possible mutagenicity. This test is important to obtaincompounds having a lower genotoxic profile, and also guides molecularchanges to obtain more safe compounds. The tests were conducted with thepreviously described compounds of general formula IA, IB, IC, ID, IE,and IIA and are shown in tables 1, 2 and 3 below.

TABLE 1 Mutagenic evaluation in Salmonella typhimurium TA100 and TA102strains in the presence and absence of metabolic activation (S9) of thecompound IA. concentration TA 100 TA 102 Compound nmol/plate +S9 −S9 +S9−S9 IA 0 129.3 ± 8.1 136.7 ± 12.4 213.5 ± 15.5 197.33 ± 16.01 7.25 154 ±14.2 (1.19) 140.5 ± 17 (1.03) 298 ± 11.3 (1.4) 263.7 ± 15 (1.33) 14.5155 ± 5.9 (1.19) 161.7 ± 11 (1.18) 320.8 ± 17 (1.5) 249.7 ± 12 (1.26) 29140.3 ± 2 (1.08) 122.7 ± 3.06 (0.9) 385 ± 21.8 (1.8) 261.3 ± 20 (1.32)56 126.3 ± 8.1 (0.97) 234 ± 39.5 (1.71) 499 ± 8.9 (2.34) 153 ± 21(0.77)* 112 133.3 ± 11 (1.03) 335 ± 15.7 (2.45) 351.2 ± 12 (1.64)* 141 ±18 (0.71)* *cell death

TABLE 2 Mutagenic evaluation in Salmonella typhimurium TA100 and TA102strains in the presence and absence of metabolic activation (S9) ofcompounds IB, IC and IIA. concentration TA 100 TA 102 COMPOUNDμmol/plate +S9 −S9 +S9 −S9 IB 0 104 ± 7.4 115 ± 13.2 219 ± 8.9 323 ±10.2 0.01 354 ± 6.9 (3.4) 171 ± 48.9 249 ± 11.7 394 ± 30 (1.22) (1.49)(1.13) 0.021 335 ± 10.8 (3.22) 200 ± 23.3 269 ± 10.6 452 ± 23 (1.55)(1.74) (1.22) 0.042 397 ± 25.9 223 ± 45.04 239 ± 22.5 502 ± 14 (1.25)(3.8) (1.94) (1.09) 0.085 395 ± 40.2 206 ± 30.2 247 ± 21.4 405 ± 24(1.34) (3.8) (1.8) (1.12) 0.17 261 ± 11 (2.5)* 165 ± 11.1 214 ± 32(0.97)* 433 ± 35.4 (1.43)* (1.54) IC 0 129 ± 8.1 179 ± 8.72 372 ± 27.5254.7 ± 14.6 0.224 165 ± 13.2 216.7 ± 10.2 272 ± 26 (0.73) 281 ± 25(1.10) (1.27) (1.21) 0.488 153 ± 13.1 224.5 ± 24.6 378 ± 6.1 286 ± 4.2(1.18) (1.25) (1.01) (1.12) 0.896 146 ± 5 (1.12) 266.3 ± 8.4 405 ± 11(1.09) 303 ± 9.2 (1.48) (1.19) 1.8 163 ± 22.5 239.7 ± 11.3 399.3 ± 40395.7 ± 9 (1.26) (1.34) (1.07) (1.55) 3.58 158.3 ± 9.3 323.7 ± 10.2 431± 17.9 387 ± 9.7 (1.22) (1.8) (1.15) (1.52) IIA 0 129.3 ± 8.14 146.7 ±12.1 372.3 ± 27.5 197.3 ± 16.1 0.98 160 ± 12.4 146.3 ± 5.1 385 ± 33.1222 ± 12 (1.12) (1.24) (0.99) (1.03) 1.96 175.7 ± 2 (1.36) 180.5 ± 7.8323 ± 5.54 241 ± 13 (1.22) (1.23) (0.87) 3.92 185.3 ± 15 179 ± 11.3423.7 ± 22 251 ± 19.2 (1.43) (1.22) (1.13) (1.27) 7.85 206 ± 13.6 172.7± 12.5 387 ± 14.4 232 ± 13.1 (1.59) (1.17) (1.03) (1.17) 15.7 354 ± 6.5(2.74) 175 ± 2.8 (1.19) 300 ± 4.9 (0.8)* 210 ± 23 (1.06) *cell death

TABLE 3 Mutagenic evaluation in Salmonella typhimurium TA100 and TA102strains in the presence and absence of metabolic activation (S9) ofcompounds ID and IE. concentration TA 100 TA 102 COMPOUND μol/plate +S9−S9 +S9 −S9 ID 0 129.3 ± 8.1 179 ± 8.7 372.3 ± 27.5 260.1 ± 11.6 0.224157 ± 13 (1.21) 228 ± 18 (1.27) 405.7 ± 13 340.3 ± 9.7 (1.09) (1.31)0.488 184.7 ± 16 239 ± 11.5 434.7 ± 21 373.5 ± 21 (1.42) (1.33) (1.16)(1.43) 0.896 216.7 ± 18 231 ± 20.2 370 ± 6 (0.99) 391.2 ± 6.3 (1.67)(1.29) (1.5) 1.8 192.7 ± 16 212.3 ± 17 397.3 ± 9.3 (1.06) 486.8 ± 20(1.49) (1.18) (1.87) 3.58 263 ± 12.3 349 ± 5.6 427.3 ± 5 (1.14) 420 ±8.6 (1.62)* (2.03) (1.94) IE 0 129.3 ± 8.1 136.7 ± 12.4 372.3 ± 27.5197.3 ± 16.1 0.12 363 ± 25.2 (2.8) 143.3 ± 5 335.3 ± 11 (0.9) 256 ± 6.9(1.29) (1.04) 0.25 478.7 ± 4.22 135.3 ± 12 295.3 ± 28 (0.8) 262 ± 19.1(3.7) (0.99) (1.32) 0.5 628 ± 40.8 136.3 ± 13 406 ± 14.1 266.7 ± 18(4.86) (0.99) (1.09) (1.35) 1 739 ± 25.4 121.5 ± 14 465 ± 10.6 265.7 ±10 (5.72) (0.89) (1.25) (1.34) 2 750 ± 13.6 177 ± 23.6 447 ± 19.8 (1.2)201 ± 4.7 (1.02)* (5.79) (1.30) *morte celular

The compound of formula IA showed mutagenicity ratios (RM) of 2.45 and2.34; in the TA100 strain, the absence of metabolic activation (112nmol/plate), and in the TA102 strain, in the presence of metabolicactivation (56 nmol/plate), respectively. At concentrations higher than56 nmol in TA102 (+S9), toxicity can be observed, with a reduction onthe number of revertants per plate (Table 01). Compound IA is an alkylderivative which has reactive methylene carbon, i.e., the carbon atomhas a positive partial charge by removal of the electron density due tothe more electronegative a moieties. The presence of this methylenecarbon promotes the attack by bionucleophiles, leading to elimination ofthe nitrate, which readily decomposes into a radical species generatinga number of detrimental effects in the DNA of the prokaryote, which doesnot have a repair system as efficient as that of eukaryotes. Further,there may occur addition of the bionucleophile into the reactivemethylene carbon, resulting in the formation of a covalent adduct,irreversibly modifying the original structure of said bionucleophile.

The compound of formula IB shows, at the concentrations used,mutagenicity in TA100 strain in the presence of metabolic activation, atall tested concentrations and with the following mutagenicity ratios(MR): 0.01 μmol (3.4); 0.021 μmol (3.22); 0.042 μmol (3.8); 0.085 μmol(3.8) e 0.17 μmol (2.5) (Table 02). Among the alkyl series tested, thatwas the one which showed the highest MR. When compound IB is comparedwith compound IA, it can be clearly seen that the latter has a lowerhindrance to attacks on the methylene carbon, facilitating the access ofthe bionucleophile. The hindrance in compound IA is higher due to thepresence of bulky moieties at positions a. This could account for thehigher mutagenicity ratio of compound IB, and allows us to predict thatmethylene compounds will be less mutagenic than ethylene compounds. Thishypothesis is better analyzed through the results of tests withcompounds ID and IE.

Compound IC did not show mutagenicity at the concentrations used,although in the test with the TA100 strain in the absence of metabolicactivation and at the concentration of 3.58 μmol/plate, it showed amutagenicity ratio of 1.8; that is, signs of mutagenicity (Table 01).When compound IA is compared with compound IC, it can be observed thatthe latter—an interphenylene derivative—has a lower mutagenicity sincethe sign has appeared only at 3.58 μmol/plate, while in compound IA,mutagenicity has occurred at 112 nmol/plate with a MR of 2.45 (TA100;−S9). Also, when the compound IC is compared with the compound IB, itcan be observed a lower mutagenicity of the interphenylene derivative.These data suggest that the aryl derivatives, that is, those having anaromatic ring bonded to the phthalimide moiety (compounds IC, ID, IE andIIA), have a lower mutagenicity than the alkyl derivatives, that is,those in which the alkyl chain is directly bonded to the phthalimidemoiety (compounds IA and IB); supporting the hypothesis that stericfactors hinder the access of the bionucleophile to the reactive site,modulating the mutagenicity of the synthesized derivatives.

The compound IIA is a sulphonamide derivative which does not have thenitrate moiety, common to all other compounds. The literature reportsthat hydroxylamine derivatives, or hydroxamic acid derivatives, showmutagenicity largely due to the major toxicophoric contribution fromthis moiety (ZHU, X. et al. Mut. Res. v. 425, pp. 153-167, 1999). For along time, the hydroxylamine moiety has been pointed out as one of themain metabolites, generated in the reduction of the nitro group,responsible for the mutagenic activity of nitro compounds (e.g.,chloramphenicol, metronidazole, and nitrofurans). Nevertheless, it hasbeen found that they are radical species formed in steps prior toreductions which generate mutagenic products rather than thehydroxylamine derivative itself (TOCHER, J. H. Gen. Pharmac. v. 28, n.4. pp. 485-487, 1997).

Thus, the compound IIA containing the hydroxylamine moiety was evaluatedin order to verify the toxicophoric contribution from this moiety in thesynthesized compound. This derivative was subsequently reacted withphthalic anhydride to obtain a phthalimide derivative.

At the concentration of 15.7 μmol/plate in the TA100 strain, withmetabolic activation, the compound IIA exhibited a mutagenicity ratio of2.74. Above 15.7 μmol/plate, there is a reduction on the number ofrevertants per cell toxicity. The possible mutagenesis of compound IIA,which occurs only in the presence of metabolic activation, could beassigned to the formation of a radical and/or oxidized derivative fromthis compound. When comparing the concentration of compound IIA used inthe test with the aryl derivatives (compounds IC, ID and IE), it can beseen that, although there is mutagenesis, it is observed only at highconcentrations, being up to 125 times higher, in number of moles, thanthat found for compound II (0.12 μmol/plate).

The compound ID showed in the TA100 strain, in the presence of metabolicactivation and at a concentration of 3.58 μmol/plate, a mutagenicityratio of 2.03; while in the absence of metabolic activity, at this sameconcentration, it showed mutagenicity signs with MR values of 1.94. At aconcentration of 1.8 μmol/plate in the absence of S9, in the TA102strain, it showed mutagenicity signs, with a MR of 1.87 (Table 03). Whencomparing the compound ID, a regioisomer of the compound IC, we observeda discrete profile of higher mutagenicity and/or a mutagenicity sign ofthe compound ID with respect to compound IC.

The compound IE, an interphenylene derivative of compound IB, in theTA100 strain and in the presence of metabolic activation, showed, aswell as compound IB, mutagenesis at concentrations of 0.12; 0.25; 0.5; 1and 2 μmol/plate, with MR values of 2.8; 3.7; 4.86; 5.72 e 5.79,respectively (Table 03). Although MR values are higher in compound IIwith respect to compound IB, the latter is at a lower molarconcentration. The need for higher concentrations for compound II toshow mutagenesis confirms, in structural terms, what had already beenobserved between compounds IA and IC: the presence of phenyl bonded tothe phthalimide moiety reduces the mutagenicity of the compounds.

When comparing compound II with compound ID, both para-substituted, wecould also confirm, as seen between compounds IA and IB, that the ethylspacing increases mutagenesis, when compared to methyl spacing. Asdiscussed previously, this factor is probably related, besides theelectron factor, mainly to the steric factor, due to the better accessby nucleophiles to the carbon a to the nitrate moiety.

When relating the obtained compounds with thalidomide and hydroxyureapatterns, we observed a sensitivity of the AMES test in responding tothe examples, when compared to HU and thalidomide. This observation,although it suggests a higher mutagenic activity of the synthesizedcompounds regarding the patterns of structural planning, can not beconclusive, and more tests are required for such statement. In addition,thalidomide, HU and synthesized compounds are structurally distinct and,due to this chemical particularity, could show a differentiatedmutagenic profile.

From the sets of results obtained in the AMES test, we could infer that:

Alkyl derivatives (compounds IA and IB) show higher mutagenicityexpressed by the average of the number of revertants/plate than arylderivatives (compounds IIA, IC and ID);

Derivatives with ethylene spacing show higher mutagenesis than methylenecompounds;

This set of results allows us to conclude that the benzyl spacing ismore suitable in order to obtain compounds with lower mutagenicity.

Further, even for mutagenic compounds, a mutagenicity ratio of 2 isconsidered low if compared to drugs used in therapeutics, such asmetronidazole, which has a MR of 14.9 when tested at 58.4 μmol in TA 100without metabolic activation (SILVA, A. T. A. et al. Mini Rev. Med.Chem., v. 5, pp. 893-914, 2005). This allows us to conclude thatalthough there are signs of mutagenicity for the compounds, it is toolow, and these results may not reflect in eukaryotic cells.

Mouse Ear Edema Assay Induced by Capsaicin

This assay is characterized by an acute inflammatory response of theear, with development of edema, and it was performed in order toevaluate the anti-inflammatory activity of the synthesized compounds.

In this assay, indomethacin was used as a control at 100 μmol/Kg, andthe phthalimide derivatives were firstly evaluated at 300 μmol/Kg viai.p. From table 4, it can be observed that compounds IC and IE show anear edema inhibition percentage higher than 64%. Compound IC showed aninhibition percentage of about 64.09% in the performed assay. The othercompounds, however, show a similar activity when compared toindomethacin (FIG. 01), taking into consideration the standard error.These results suggest that the synthesized compounds showanti-inflammatory activity in the acute phase, probably due to theinhibition of the cytokine TNFα, since it is known that phthalimidederivatives have this activity.

TABLE 4 Ear edema assay induced by capsaicin (i.p.) + Compound CompoundCompound Compound Compound Compound Control IA IB IC IIA ID IE N 5 5 5 55 5 5 Average — 53.75 46.55 64.09 49.24 37.18 76.58 inhibition % EPM —8.66 3.49 6.44 10.58 19.38 5.32 * Animals showed higher values thancontrols

Peritonitis Assay

In a second assay, the number of total leukocytes (10⁶/mL) was evaluatedin order to evaluate the ability to inhibit their infiltration in theinflammatory process. All phthalimide derivatives showed inhibitionactivity of the leukocyte infiltrate (FIG. 2; Table 5) with a similaractivity profile, considering the standard error. These resultsdemonstrate the anti-inflammatory potential of these compounds.

TABLE 5 Results of the peritonitis assay Compound Compound CompoundCompound Compound Compound + Control IA IB IC IIA ID IE N 3 3 3 3 3 3 3Inhibition % 12.5 51.25* 42.85 28.57 32.14 26.25 Cell 5.6 4.9 2.73 3.24.0 3.8 4.13 Number avg (×106) EPM 0.20 0.7 1.0 0.8 0.30 0.2 1.16 # Theanimal has bleed • P < 0.05 (Student's T test)

Abdominal Writhing Induced by Acetic Acid

In order to evaluate the peripheral analgesic activity of the compounds,an abdominal writhing assay induced by acetic acid (Table 6) was carriedout. In this assay, we could infer that compound ID has an importantanalgesic activity, inhibiting abdominal writhing by 66%. Othercompounds, such as IA, IIA and IE, also have significant inhibitions ofthe abdominal writhing induced by acetic acid, demonstrating theanalgesic potential of these compounds. The analgesic activity may berelated to the ability of these compounds to inhibit the cytokine TNFα,since it is known that this would be one of the mechanisms that explainthe analgesia of molecules such as thalidomide.

TABLE 6 Abdominal writhing assay induced by acetic acid CompoundCompound Compound Compound + Control IA IB Compound IIA ID IE Average47.54 28.6 39.2 30.4 16.2 Inhibition % — 39.83 23.22 36.05 66.03 33.95EPM 3.39 9.25 8.65 7.83 5.24 6.60

Assays for Evaluating the Increase of Gamma-Globin by PCR in K562 CellCulture

From results obtained in the evaluation of the gene expression inducedby compounds in a culture of K562 erythroleukemic cells, and quantifiedby Real Time PCR, we can conclude that:

Compound II has activity in this model, increasing the gene expressionof gamma-globin, in the presence or absence of hemin;

In the presence of hemin, compound II did not show a significantlyhigher activity than in the absence of hemin, when compared to thecontrol (FIG. 3);

Apparently, compound II has an effect on the expression of gamma-globinat low concentration (5 μM e 30 μM);

The compound II showed high percentages of cell viability (higher than90%) in assays with and without hemin, demonstrating the absence oftoxic effects at the concentrations used;

Compound IIA showed higher activity than the control in the expressionof gamma-globin (FIG. 4).

The increase on the expression of gamma-globin induced by compound IIAis higher than the increase provided by compound II, suggesting thatcompound IIA is more efficient in increasing the gene expression ofgamma-globin.

When comparing the compound IIA with HU data in the literature, it wasobserved that the compound IIA shows activity at 5 μM in 48 hours,whereas for HU to produce a comparable activity, it is used at 10 μM inthe same time.

The cell viability at 0 h was 97%, and this pattern was maintainedduring the realization of the assay, demonstrating the absence oftoxicity of the compound IIA (FIG. 5).

Methodologies Used for Pharmacological Assays:

Procedures for Evaluating the Mutagenic Activity (AMES Test)

The procedure was firstly developed by (MARON, D. M. and AMES, B. N.Mut. Res. v. 113, pp. 173-215, 1983)

Strains Used in the Assay:

There are many genetically modified strains of Salmonella typhimurium inorder to detect a prevalent type of mutation, which include: TA97, TA98,TA100 and TA102. TA100 and TA102 detect mutations which cause base pairsubstitutions, while TA 98 and TA 97 detect changes where there is a gapin the DNA reading frame (MARON & AMES, 1983).

For this assay, Salmonella typhimurium TA100 and TA102 strains from themutagenicity laboratory of the “Faculdade de CiênciasFarmacêuticas—UNESP Araraquara” were used. Such strains have thefollowing characteristics: (AMES, 1983)

-   -   1—Are auxotrophic with respect to histidine;    -   2—Have various mutations in the histidine operon, which are        target for reverse mutation;    -   3—Detect many mutagenic agents which cause shift in the DNA        reading frame, which restore the correct reading frame for        histidine synthesis;    -   4—Mutation in hisG46 gene, in the reading frame of the hisG        gene, which encodes the first enzyme for histidine synthesis, TA        100-specific;    -   5—Mutation in gene hisD3052, constituted by 8 repeated residues        of -GC-, near the shift mutation site in the reading frame of        the hisD gene, which encodes the TA 98-specific histidinol        dehydrogenase enzyme;    -   6—Mutation (rfa), which causes partial loss of the        lipopolysaccharide barrier, increasing the permeability of the        bacterial cell wall, facilitating the diffusion of large        molecules into the cell;    -   7—Mutation (urvB), which causes damage in the repair system by        excision, resulting in an increase on the detection sensitivity        of various mutagenic agents. It also causes the bacterium to        become dependant on biotin to grow;    -   8—Plasmid pKM101, which enhances the resistance to ampicillin,        and also increases the spontaneous and chemical mutagenesis by        stimulating the error-prone DNA repair system.

Maintenance and Storage of Strains

Salmonella typhimurium strains were stored in a freezer at −80° C., inflasks for freezing with 0.9 mL of culture and 0.1 mL of DMSO as acryoprotector agent, so as to maintain all their genetic characteristicsunchanged.

Before freezing, all strains had their genotypes confirmed (histidineauxotrophy, rfa mutation, pKM101 plasmid, uvrb deletion, and spontaneousreversion rate).

Preparation of Culture Media and Solutions

Vogel-Bonner Medium E (VB)

0.25 g of magnesium sulphate, 2.5 g of citric acid, 12.5 g of dibasicpotassium phosphate, and 4.375 g of sodium and ammonium phosphate weredissolved into 16.75 mL of distilled water at 45° C. (amounts enough for25 mL of VB solution). The solution was sterilized in an autoclave for15 minutes at 121° C.

40% Glucose

50 mL of a 40% glucose solution were prepared, which was sterilized inan autoclave for 15 minutes at 121° C.

Glycosylated Minimum Agar (GMA)

7.5 g of agar was dissolved into 465 mL of distilled water, and then thesolution was sterilized in an autoclave for 15 minutes at 121° C.

Subsequently, a sterile laminar flow, 10 mL of VB, and 25 mL of 40%glucose were added.

Top Agar

0.5 g of sodium chloride and 0.6 g of agar were dissolved into 100 mL ofdistilled water. The solution was sterilized in an autoclave for 15minutes at 121° C.

0.05 mM biotin/histidine solution (10 mL/100 mL of top agar)

0.00123 g of biotin and 0.00096 g of histidine were dissolved into 10 mLof distilled water. The solution was sterilized in an autoclave for 15minutes at 121° C.

Oxoid nutrient broth n. 2.

0.75 g of Oxoid medium was dissolved into 30 mL of distilled water. Thesolution was sterilized in an autoclave for 15 minutes at 121° C.

Positive and Negative Controls

The negative control is the solvent used to dissolve the sample, usingas a standard volume, the highest volume of the tested sample 100 μL,which is also the amount that is required to dissolve the maximum usedconcentration of the drug.

The positive controls are mutagenic compounds specific for each strainand test condition, with 25 μL/plate of sodium azide (1.25 μg/plate) and100 μL of mitomycin (0.5 μg/plate) being the controls for TA100 andTA102, respectively, in the absence of metabolic activation. For assayswith metabolic activation, the positive control for TA100 is 50 μL2-antramine (1.25 μg/plate) and for TA102 it is 50 μL 2-aminofluorene(1.25 μg/plate).

Assay Procedure without Metabolic Activation System (−S9)

The preincubation method was used.

1^(th) Day

All solutions and culture media previously described were prepared. Inlaminar flow, 10 mL of VB solution and 25 mL of 40% glucose solution(previously prepared) were added to the sterile material (GMA), followedby homogenization, and about 25 mL of AMG were distributed to eachplate.

The GMA distributed to the plates was left under rest for 48 hours in anoven at 37° C. for subsequent use.

2^(th) Day

In laminar flow, Salmonella typhimurium (TA100 and TA 102) strains wereinoculated individually with a platinum loop, in the respective nutrientbroths and maintained at 37° C., under constant stirring (160 rpm)during 14 hours, in order to achieve a density of 1 to 2×10⁹bacteria/mL.

3^(th) Day

Different concentrations of the compounds were added into 100 μL of 0.2Mphosphate buffer pH 7.4 (or 500 μL of the mixture S9 in metabolicactivation assays) and incubated for 20-30 minutes at 37° C. Solutionscontaining the compounds had DMSO as the solvent. Thereafter, 2 mL oftop agar supplemented with traces of histidine and biotin was added,homogenized and plated in glycosylated minimum medium. Aftersolidification of the top agar, the plates were incubated for 48 hoursat a 37° C. Then, the counting of the number of revertant colonies perplate was performed. All tested concentrations, positive and negativecontrols were run in triplicate.

5^(th) Day

After 48 hours, the revertant colonies were counted manually, and theprotoCOL Colony Counter Version 3.15.630 (1998-2001) SYNBIOSIS LTDsystem was used for the positive control.

Evaluating and Interpreting the Results

The final data obtained from the assay was analyzed using thestatistical software Salanal (Salmonella Assay Analysis) version 1.0from the Research Triangle Institute, RTP, North Carolina, USA. Suchsoftware allows the dose-response effect to be evaluated by means ofanalysis of variance (ANOVA—test F) computations between the measurementof the number of revertants at the different tested concentrations(doses) and the negative control, followed by a linear regression. Thesoftware model chosen for analyzing the data was Bernstein's (BERNSTEIN,L. et al. Mutat. Res. v. 97, p. 267-281, 1982.). The slope of thestraight line from the linear portion of the dose-response curve is alsoprovided by this software and corresponds to the number of revertantsinduced per measurement unit of the analyzed sample.

From the results, the mutagenicity ratio (MR) was computed for eachanalyzed dose from each compound. MR is given by the following equation:MR=average number of revertants Per test plate(spontaneous+inducedrevertants)

average number of revertants per plate of the negative control(spontaneous revertants)

The spontaneous growing means that the number of revertants whichdeveloped on the plate, regardless of being induced or not, whereinvalues higher or equal to 2 are considered as a positive response(VALENT, G. V. et al. Env. Toxicol. Water Quality. v. 8, p. 371-381,1993.).

Assay Procedure with Metabolic Activation System (+S9)

The mutagenicity test with metabolic activation system was performedwith a microsomal fraction S9 (S9 mix) prepared from a liverhomogenizate from Sprague Dawley rats, previously treated with Aroclor1254, acquired in the freeze-dried form.

50 mL of S9 mix were prepared using the following solutions shown inTable 7 below:

TABLE 7 Solutions used for the preparation of S9 mix. Sterile water19.75 mL Phosphate buffer 0.2M 25 mL NADP 0.1M 2 mL (freezer) G-6-P 1M250 μL (refrigerator) MgCl 0.4M 500 μL (refrigerator) KCl 1.65M 500 μLS9 Fraction Dissolved into 2 mL of sterile miliQ waterThe procedure for this assay is the same, however, instead of thebuffer, 500 μL of the S9 mixture should be added.

The S9 mixture has a viability of 4 hours from preparation when put onice. The plates are then incubated for 48 hours at 37° C. After therequired time has elapsed, the counting of the revertant colonies wascarried out. All tested concentrations, positive and negative controlswere run in triplicate.

Procedures for Assay in K562 Cell Culture

The human leukemia cell line K562 ATCC (American Type CultureCollection), Philadelphia, Pa., USA was used. The cells were cultured inDMEM medium (Dulbecco's

Modified Eagle Medium, Invitrogen, USA) containing 10% of fetal bovineserum and glutamine. The cells were maintained at 37° C. under a 5% CO₂atmosphere. For the experiments, the cells were incubated at a densityof 1×10⁵ cells/mL. In order to carry out the culture with hemin (30 uM),it was added 72 hours before the beginning of the experiment with thedesired compound.

The 0-hour time consisted in removing non-treated K562 cells. From thispoint, the respective compounds were added at the desired concentrations(5, 30, 60 and 100 uM), the cells were then maintained for 7 days underculture, without a new addition of any compound or substitution of theculture medium. Cell collections were performed at the following times:0, 24, 48, 72 and 96 hours. The morphology of the cells was analyzed atthese points through cytospin slides stained with Leishman and the cellviability was performed by staining with trypan blue in a Neuberger'schamber.

RNA Extraction

The extraction method with TRIzol reagent (Gibco-BRL, Gaithersburg, Md.)according to the manufacturer's instructions was used in order to obtainthe RNA from K562. The sample containing K562 and TRIzol was incubatedfor 5 minutes at room temperature in order to achieve completedissociation of the nucleoproteic complexes, 200 μL of chloroform(CHCl₃) was added and thoroughly stirred, and incubation was againperformed by 5 minutes at room temperature. After centrifugation for 15minutes at 19,000 g at a temperature of 4° C., the supernatant wasobtained and stored in another tube, proceeding immediately to the stepof precipitation with 500 μL of cold isopropanol. After homogenization,a new incubation was carried out for 10 minutes at room temperature,followed by centrifugation for 10 minutes at 19,000 g at 4° C. Thesupernatant was disposed of and 800 μL of 70% cold ethanol was added tothe precipitate, with a new centrifugation being carried out for 5minutes at 14,000 at 4° C. Finally, the supernatant was disposed of andthe RNA precipitate was left to dry for 10 minutes at room temperature,and then resuspended in sterile water containing diethyl pyrocarbonate(DEPC) and incubated at 55° C. for 10 minutes and subsequently put onice for total solubilization of the RNA.

The sample was checked as to its integrity by electrophoresis on a 1.2%denaturing agarose gel. The samples having a suitable amount of RNAshowed integrity on both ribosomal subunits: 18S e 28S. Afterelectrophoresis, the RNA samples were stored in a freezer at −80° C.

Complementary DNA (cDNA) Synthesis

The RNA samples obtained were subjected to the complementary DNA (cDNA)synthesis using the Superscript III RTTM kit (Invitrogen, LifeTechnologies). After reading in a spectrophotometer (GeneQuant-Pharmacia, USA) and quantification, 3 μg of RNA were treated withthe enzyme DNase I (Invitrogen, Life Technologies), for removal ofcontaminant DNA. 1.0 μL of 1 u/μL DNase I, 1.0 μL of 10× DNase IReaction Buffer (200 mM Tris-HCl, 20 mM MgCl2, 500 mM KCl2) and watersufficient for a final volume of 10.0 μL of reaction were added. Thereaction was carried out for 15 minutes at room temperature and stoppedwith 1.0 μL of 25 mM EDTA, and incubated for 10 minutes at 65° C.

For cDNA synthesis, 1.0 μL of 50 μM oligo (dT) 20 and 1.0 μL of 10 mMdNTP's were then added. The samples were incubated for 5 minutes at 65°C., followed by 1 minute at 4° C. To each sample, 10.0 μL of thefollowing reaction mixture were added: 2 μL of 10×RT buffer, 4.0 μL of25 mM MgCl2, 2.0 μL of 0.1 M DTT, 1.0 μL of 40 U/μL RNase OUTTM and 1.0μL of 200 U/μL Superscript III RTTM. The reaction occurred for 50minutes at 50° C., followed by 5 minutes at 85° C. Thereafter, 1.0 μL of2 U/μL E. coli RNase H was added for 20 minutes at 37° C.

Verification of Complementary DNA Synthesis

The verification of cDNA synthesis was made by means of PCR foramplification of the beta-actin (BAC) gene. Reactions were carried outwith: 5.0 μL of 10×PCR buffer (20 mM Tris-HCl, 500 mM KCl), 1.5 μL of 50mM MgCl2, 1.0 μL of 10 mM dNTP's, 1.0 μL of 10 mM of BACF primer(5″-AAGAGATGGCCACGGCTGCT-3″), 1.0 μL of 10 mM of BACR primer(5″-TCGCTCCAACCGACTGCTGT-3″), 0.5 μL of Taq DNA polymerase, 1.0 μL ofcDNA and 39 μL of water, for a final volume of 50 μL. The program wasstarted for 2 minutes at 94° C., followed by 35 cycles: 94° C./30seconds-58° C./45 seconds-72° C./40 seconds, being terminated by 72°C./7 minutes. The products were subjected to electrophoresis on 1%agarose gel to verify the amplification of 640 pb.

Design of Primers for Quantitative Real-Time PCR Reaction

The primers used in Quantitative Real-Time PCR reactions were designedwith the software “Primer Express” (Applied Biosystems), analyzed in theprogram Blast (www.ncbi.nlm.nih.gov/blast) to verify the conditions forformation of structures, such as hairpins and dimers.

Standardizations Required for Quantitative Real-Time PCR

Primer Concentration

The optimum concentration of primer to be used in quantitative real-timePCR should be sufficiently low to allow duplication of all copies of thegene present in the sample. Using the same amount of sample, reactionscontaining each of the primers (sense and antisense) were performed atthe final concentration of 150 nM, 300 nM, 600 nM e 900 nM. The cycle inwhich fluorescence is detected above the established threshold is calledthreshold cycle or Tc. Since the same amount of sample was used in allreactions, the Tc should not change. If the increase on the primerconcentration caused a reduction in Tc, so the amount of this reagent inthe reaction was still insufficient. Thus, the optimum concentrationchosen was the minimum, associated with the lower Tc.

The sequence and length of the amplified fragments from each primer pairused in the amplification of the genes studied in the quantitativereal-time PCR technique is shown in table 8.

TABLE 8 Sequence and length of the amplified fragments Length of Primerthe amplified Gene Sequence fragment Gamma-Glob-5′-CCAGCTGAGTGAACTGCACTGT-3′ 81 bp F 5′-ACGGTCACCAGCACATTTCC-3′Gamma-Glob- R β-actin-F 5′-AGGCCAACCGCGAGAAG-3′ 79 bp β-actin-R5′-ACAGCCTGGATAGCAACGTACA-3′ GAPDH-F 5′-GCACCGTCAAGGCTGAGAAC-3′ 89 bpGAPDH-R 5′-CCACTTGATTTTGGAGGGATCT-3′

The primer concentrations used in the amplification of the studied genesand the amplification efficiency obtained is shown in Table 9. Theconcentrations were defined by the amplification efficiency generatedunder the tested conditions.

TABLE 9 Primer concentrations Primer Used concentration Primerefficiency Gamma-glob 150 nM 100% β-actin 300 nM 100% GAPDH 300 nM 100%

Reaction Efficiency

In order that the real-time PCR reaction is reliable and reproducible,optimum reaction conditions are required, i.e., the amplifications mustshow 100% amplification efficiency at every cycle, occurring sampleduplication. The amplification efficiency is obtained from formula 10(−1/slope), wherein slope means the slope value of the curve.Optimization occurs using the optimum primer concentration with 7 knownsample amounts, in logarithmic scale: 2 ng (2×100), 6.32 ng (2×100.5),20 ng (2×10¹), 63.2 ng (2×101.5), 120 ng, 200 ng (2×102) e 632 ng(2×102.5). The results are used to construct a standard curve Tc versussample amount.

Quantitative Real-Time PCR

After reading in a spectrophotometer (Gene Quant-Pharmacia, USA) andquantification, cDNA aliquots were used as template in quantitativereal-time PCR reactions. The technique consists in optically monitoringthe fluorescence emitted during the PCR reaction, by means of thebinding of a specific probe or dye to the newly synthesized strand.

The reactions, always run in duplicate, were performed using the reagentSYBERGreen PCR Master Mix® (Applied Biosystems), which in addition tocontaining all reagents required for PCR (dNTP's, MgCL2, buffer, TaqAmpli-Gold), also contains the SYBERGreen dye, a double-strandedintercalating agent necessary for the detection of the reaction fromcycle to cycle. Further, cDNA samples and specific primers for theanalyzed gene were used.

The real-time amplification detection was performed in the apparatus ABI5700 Sequence Detector System® (Applied Biosystems) in fluorescenceversus cycle number graphs. The higher the expression of a gene, thatis, the more copies there are at the beginning of the reaction, theearlier will occur amplification, and hence, the lower will be the Tc.

The reactions carried out contained 12.5 μL of the reagent SYBERGreenPCR Master Mix®, 25 ng of cDNA sample and the optimum determined primerconcentration, making up a final volume of 25 μL. In all cases, negativecontrols were made containing sterile water in place of the sample. Thereactions were prepared in 96-well plates (Sorenson, BioScience Inc)with plastic caps which allow the passage of light. The program wasstarted at 95° C./10 minutes, followed by 45 cycles: 95° C./15seconds-60° C./1 minute. At the end of a normal amplification, adegradation step is added during which the temperature rises graduallyfrom 60° C. to 95° C. As the products generated by PCR denaturate withthe temperature rise, the florescence signal of SYBR Green decreases.The resulting graph allows verifying if there is one or more PCRproducts present in each reaction, due to melting temperaturedifferences between amplified PCR products, this difference being causedby the number and composition of bases of each product.

Analysis of Real-Time Data

The expression of the genes of interest was determined in a relativeway, being normalized with respect to genes called calibrators; in thisstudy, we used β-actin and GAPDH, which are genes whose expression issupposedly constitutive, that is, they show little variation betweenvarious conditions. However, some studies have been demonstrating thatthe expression of these genes may change substantially. From the Tcvalues obtained, the arithmetic mean of Tc duplicates was computed.Subsequently, the amount of expression (Q) was obtained by means of theformula Q=EdeltaTc, where E=reaction efficiency and delta Tc=lowest Tcobserved−Tc of the sample. Thus, the expression was related with thesample which exhibited the highest expression (Lowest Tc observed),which received a value Q=1. The Q values of the calibrator genes of eachsample were subjected to the Gnorm program, which computes the geometricmean between the same, which value is called sample NormalizationFactor. The normalized expression of a given gene of interest in acertain sample is given by the ration between value Q of the gene ofinterest of the sample and the sample Normalization Factor. The obtaineddata is expressed in arbitrary units or absolute expression value.

Abdominal Writhing Assay Induced by Acetic Acid

The antinociceptive profile was evaluated through the abdominal writhingassay induced by acetic acid in mice. In this assay, Swiss mice fromboth genders were used, weighing from 21 to 28 grams, fasted for a timeperiod of about 8 hours. The test substance was administered orally andhad, as a carrier, 5% gum arabic. After 1 hour, the administration ofacetic acid 0.1N (0.1 mL/10 g weight) was performed in the peritonealcavity of the animals. Ten minutes after the injection of acetic acid,writhings were counted during 20 minutes. Controls were made for thecarrier (gum arabic) and it does not show pharmacological activity.

Mouse Ear Edema Assay Induced by Capsaicin

This assay was performed using Swiss mice from both genders weighingfrom 18 to 30 grams. The animals were fasted for 8 hours with freeaccess to water. This assay consists in locally administering (rightear) 20 μl of a capsaicin solution (250 μg/ear) diluted in acetone, 1hour after the i.p. administration (diluted in 0.5% gum arabic). Theleft ear (control) received the carrier in which capsaicin was diluted(acetone) and the right ear received capsaicin. This assay ischaracterized by an acute inflammatory response of the ear, withdevelopment of edema. The animals were sacrificed and their ears wereweighted to obtain the inflammation index. A biopsy (8 mm in diameter)of the ear was carried out. Next, the weights of the inflamed ears werecompared against the weights of the contralateral ear (control ear)which were not treated with the phlogistic agent. The edema inhibitionpercentage was calculated by subtracting the ear treated with thecarrier by that treated with capsaicin from each group of animalstreated with the test substances, and then it was divided by thedifference between the groups of the irritating agents and the controlgroups. The result was subtracted by 1 and multiplied by 100, beingshown in Table 4.

Peritonitis Assay

The mice were treated with the substances being analyzed or carrier andafter 1 h of oral administration, they were simultaneously subjected tothe peritonitis assay, by administering intraperitoneally 1 ml of a 3%thioglycolate solution. 4 h after the administration of thioglycolate,the peritoneal cavity was washed with 3 ml of a HANKS solution (Balancedsalt solution, free of Ca²⁺ and Mg²⁺). Next, the peritoneal wash wasanalyzed and the total counting of white blood cells was made in aNewbauer's chamber under an optical microscope with a 40× objective.Results are shown in Table 5.

Statistical Analysis

The significance levels between experimental groups and the control weregenerated using the Student's T Test. The values were consideredsignificant when *P≦0.05. Results were expressed as mean±standard errorof the mean, as indicated in the legends of figures.

Citotoxicity Assay in Mouse Peritoneal Macrophages

The determination of cell viability (FIG. 5) was performed by suspendingperitoneal macrophages in a RPMI solution, set at a concentration of5×10⁶ cells/mL, 100 μL of which were added to each cavity of 96-cavitytissue culture plates, being incubated with drugs and prodrugs atconcentrations of 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸mM during 24 hours at 37°C. and 5% of CO₂. The absorbance reading was performed in a UV/Visiblespectrophotometer at 540 nm with reference filter at 620 nm (MicroplateReader-Model 550-BIORAD).

Obtainment and Culture of Peritoneal Exudate Cells:

Mice were previously inoculated with 3.0 mL of 3% thioglycolateintraperitoneally in order to stimulate the macrophages of this cavity.After 3 days of stimulation, the animals were sacrificed and theperitoneal macrophages collected. The cells were then washed from 2 to 3times per centrifugation at 358 g (Centrifuge Fanem Excelsa II 206 MP)during 5 minutes in sterile PBS and, then, resuspended in 1 mL ofRPMI-1640 for counting in a Neubauer's chamber. After counting, theconcentration was set at 5×10⁶ cells/ml and these cells were distributedto disposable sterile plates with 96 cavities. The plates thuscontaining the suitable concentration of cells were taken to incubationfor 24 hours in a oven at 37° C., containing 95% of moisture and 5% ofCO₂ in the presence of the anti-inflammatory agents and taurinederivatives at the respective concentrations: 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵ and10⁻⁴ mM or even in the presence of the RPMI-1640 medium only. LPS wasused as a positive control and the RPMI-1640 medium as a cell control.The culture supernatants, upon the end of the incubation period, werecollected in order to determine the nitric oxide levels thereof. TheRPMI-1640 used during this whole process was supplemented with 2 mMI-glutamine, penicillin (100 U/ml), streptomycin (100 ug/ml), 5% ofbovine fetal serum and 2-mercaptoethanol 5×10⁻² M (RPMI-1640-C).

Nitric Oxide Dosage

After obtaining the supernatants from macrophage cultures, as describedabove, the concentration of nitric oxide was evaluated. This evaluationwas made by measuring the concentration of accumulated nitrite (a stabledegradation product of nitric oxide) through a diazotization reactionwith the Griess reagent (1% sulfanilamide, 0.1% naphthylenediaminedihydrochloride, in 5% of phosphoric acid), according to the method ofGreen et al. (1982). To do so, 50 μl of the culture supernatant wasincubated with the same volume o Griess reagent at room temperature (10minutes) and, thereafter, absorbances were measured at 550 nm in a ELISAreader (Microplate Reader-Model 550-BIORAD). Nitrite concentrations wereobtained from a standard curve previously prepared with known NaNO₂molar concentrations. The tests were run in triplicate and the valuesexpressed in micromole of NO²⁻/5×10⁵ cells. Results are shown in FIG. 6.

1. Compound characterized by being of formula (IE):